Project Summary/Abstract Proteasome inhibitors can kill Plasmodium spp.. However, lack of malaria-specific proteasome inhibitors that spare human proteasomes has so far precluded treating malaria with drugs like bortezomib, carfilzomib and ixazomib, which have significant toxicity. There is an urgent need to develop malaria-specific proteasome inhibitors. Our past work, including substrate profiling, enzymology, structure-guided rational design and high throughput screening, led to discovery of the first species-selective proteasome inhibitors (active against mycobacterial proteasomes but not human proteasomes) as well as highly isoform-selective proteasome inhibitors (active against the human immunoproteasome but not the human constitutive proteasome). Informed by those experiences, we collaborated with Dr. Laura Kirkman, parasitologist and co-PI, to identify a novel class of compounds that kill P. falciparum in vitro but spare mammalian cells. These compounds inhibit the P. falciparum proteasome (Pf20S) ?5 subunit potently, noncovalently and noncompetitively. The chemophore, subunit specificity, noncovalent reactivity and noncompetitive mode of inhibition of these compounds are distinctive compared to a Pf20S ?2 inhibitor recently reported by Bogyo?s team, thereby offering an independent shot on goal against a well validated target, an opportunity to overcome resistance to one agent by using the other, and the possibility of synergistic results from using both, if they each lead to drugs. Our inhibitors are highly potent in inhibiting growth of P. falciparum at erythrocytic, liver, and gametocyte stages and are equally effective against P. falciparum isolates that are sensitive or resistant to current drugs. We have formed a novel organizational structure to pool the resources of the Lin chemistry/enzymology lab and the Kirkman parasitology lab (for which this support is requested) with the expertise of two major drug companies, each donating services through not-for-profit organizations (Tri-I Therapeutics Discovery Institute and its partner, Takeda Pharmaceuticals, and Tres Cantos Open Lab Foundation and its partner, GlaxoSmithKline). We now aim to continue our team approach to advance the development of malarial proteasome inhibitors as antimalarial drugs by improving their selectivity, specificity and pharmaceutical properties. Specific Aim 1 optimizes the hit compound series through rational design and concise and parallel synthesis, then determines their in vitro potency and selectivity, tests their anti-Plasmodium potency at erythrocytic, gametocytic, and liver stages, and improves their in vitro and in vivo pharmaceutical properties. Aim 2 investigates the mechanism of resistance to Pf20S inhibitors and the synergy of Pf20S inhibitors with other anti-malarial drugs.